78 Association of viable Mycobacterium leprae with Type 1 reaction ...

Association of viable Mycobacterium leprae with

Type 1 reaction in leprosy

MRUDULA PRAKASH SAVE*, ANJU RAJARAM

DIGHE*, MOHAN NATRAJAN**

& VANAJA PRABHAKARAN SHETTY*

*The Foundation for Medical Research, Worli, Mumbai- 400 018,

India

**National JALMA Institute for Leprosy and other Mycobacterial

Diseases (NJIL & OMD). Tajganj, Agra- 282001, India

([email protected])

Accepted for publication 3 November 2015

Summary

The working hypothesis is that, viable Mycobacterium leprae (M. leprae) play a

crucial role in the precipitation of Type 1 reaction (T1R) in leprosy.

Material and Methods: A total of 165 new multibacillary patients were studied.

To demonstrate presence of viable M. leprae in reactional lesion (T1Rþ ), three tests

were used concurrently viz. growth in the mouse foot pad (MFP), immunohisto-

chemical detection of M. leprae secretory protein Ag85, and 16s rRNA - using in situ

RT- PCR. Mirror biopsies and non reactional lesions served as controls (T1R2).

Findings: A significantly higher proportion of lesion biopsy homogenates obtained

at onset, from T1R(þ) cases have shown unequivocal growth in MFP, proving the

presence of viable bacteria, as compared to T1R(2 ) (P , 0·005). In contrast, few

Mirror biopsies were positive in both T1R(þ) and T1R(2 ).

With respect to Ag85, while the overall positivity was higher in T1R(þ) (74%),

however the intensity of staining (Grade $ 2þ) was disproportionately higher in

T1R(þ) BT–BB lesions 11/20 (55%).

In the rebiopsies obtained during a repeat episode of T1R, Ag 85 as well as 16s

rRNA, positivity (62% & 100%) was higher in T1R(þ).

It is inferred therefore ‘viable’ bacteria are an essential component in T1R and

difference in the quality of bacilli, not the quantity or the ratio of dead to viable play a

role in the precipitation of T1R.

In conclusion, the findings show that ‘metabolically active’ M. leprae is a

component/prerequisite and the secretory protein Ag 85, might be the trigger for

precipitation of T1R.

Keywords: Leprosy, Viable M. leprae, Type 1 reaction, Mouse Foot Pad, Ag85, 16s r-RNA,in situ RT-PCR

Correspondence to: Vanaja P Shetty, The Foundation for Medical Research, Worli, Mumbai- 400 018, India(Tel: þ91-022-24934989; Fax: þ91-022-24932876; e-mail: [email protected])

Lepr Rev (2016) 87, 78–92

78 0305-7518/16/064053+15 $1.00 q Lepra

Introduction

The two main types of reaction Type 1 (T1R) or reversal reaction and Type 2 (T2R) or

Erythema Nodosum Leprosum (ENL), are important causes of morbidity and nerve function

impairments (NFI) in leprosy.1 T1R is more frequent, afflicting , 30% of borderline group

[i.e. Borderline Tuberculoid (BT) to Borderline Lepromatous (BL)] of leprosy patients.2

Reactions and the associated NFI are therapeutically managed with immunosuppressive and

anti-inflammatory drugs such as corticosteroids.3 – 5

Notable features of T1R are:

(a) Symptoms and signs may be confined to the skin, the nerve(s), or may occur in both

(b) It is common in borderline (BT-BL) type of leprosy: 25 to 50% patients experience

T1R sometime during the course of disease, before, during and after treatment.3,5

(c) A previous episode of reaction places a patient at higher risk of a repeat episode.5,6

(d) That it has an immunological basis, but the trigger is largely unknown.7

In the present study, we propose that metabolically active Mycobacterium leprae (M. leprae)

in a skin or nerve lesion(s) are a trigger, that the threshold of cell mediated immune reactivity to

whole M. leprae is decreased in the presence of M. leprae antigens. In brief, that incomplete

killing or refractoriness to treatment or persistence of M. leprae increases the risk of reaction.

The proposal finds support from an earlier study conducted in this laboratory on skin

biopsies in 25 smear negative BT patients manifesting late reversal reaction. The presence of

viable M. leprae was demonstrated by growth in the foot pads of non immnuno-suppressed

Swiss white (S/W) mice in 12 patients (48%). Secondly, the lesions showing sub-clinical T1R

(histopathology only) scored higher in the Mouse Foot Pad (MFP) test (7/12 ¼ 58%) as

against non-reaction cases (5/13 ¼ 39%) suggesting that viable M. leprae in a given site may

be involved in the induction of T1R.8

The present study elaborates on the earlier findings using additional tests for bacterial viability.

Materials and methods

STUDY POPULATION/ELIGIBILITY CRITERIA

Newly detected leprosy patients of either sex, aged between 15–60 years, with BT, BB or BL

type of disease (Ridley-Jopling scale), and requiring a full course of World Health

Organization Multibacillary – Multi Drug Therapy (WHO MB-MDT).

Demonstration of viable M. leprae in a skin or nerve lesion was done using three tests:

A. immunohistochemical detection of M. leprae secretory protein Ag 85;

B. detection of M. leprae specific 16s rRNA using In situ reverse transcriptase polymerase

chain reaction (in situ RT- PCR)

C. Bacterial multiplication using Mouse Foot pad (MFP) technique.

Rationale for choosing a combination of tests for viability

(a) Ag 85, is a 30kDA protein, characterised as mycolyl transferase, secreted during

growth and multiplication of M. leprae.9,10 Hence presence of Ag 85 in the lesion

suggests actively growing M. leprae.

Type 1 reaction and viable Mycobacterium leprae 79

(b) RNA detection is a highly sensitive molecular biological tool. The presence

of/localization of M. leprae specific 16s rRNA will provide further confirmation.11,12

(c) A positive MFP test, which is the gold standard for determining M. leprae viability,13

will clinch the findings in (a) and (b) above

TREATMENT FOR LEPROSY

All study patients were treated with 12 months MB-MDT regimen as per the National

Leprosy Eradication Programme WHO/NLEP guidelines.2

Treatment for reaction/neuritis: Patients presenting with, or developing (T1R) in the skin or

nerve were treated with corticosteroids 40 mg (1mg/kg body weight) tapered to 5mg over 12

to 16 weeks.2

Determination of sample size: The sample size was calculated on the basis of ‘sample size for

comparison of proportions’.14 Based on the fact that around 45% of BT-BL patients present

with or develop clinical T1R, at 95% confidence interval, a sample of ,150 BT-BL cases

was required to be recruited into the study.

Recruitment and monitoring of patients: A total of 165, newly diagnosed MB patients

fulfilling the Ridley-Jopling classification as BT, BB and BL were recruited in the study.15

They were investigated at the baseline as described below and were followed up/monitored

for a minimum period of 18 months thereafter.

Statistical analysis: Analysis was performed using SPSS version 19.0. The significance of

association was tested using Chi-square test. The 95% confidence interval was calculated.

A: Clinical investigations:

Detailed history taking and systematic physical and neurological examination with a

emphasis on signs and symptoms of reaction/neuritis and deformity (graded using WHO

deformity grades) were recorded using a standard protocol created at the Foundation for

Medical Research (FMR).

ETHICAL CLEARANCE

The study followed International Ethical Guidelines for Biomedical Research involving

human subjects (CIOMS/WHO, 1993). This study received ethical clearance from the

Ethics Committee of the Foundation for Medical Research. This included permission for

skin and nerve biopsies. Written consent was obtained from individual study subjects

before inclusion in the study using a standard consent form. No financial incentives were

given to the patients. Travel expenses were reimbursed to the patients on the occasion.

Committee for the Purpose of Control and Supervision of Experiments on Animals

(CPCSEA) cleared the use of animals in the study. The Foundation for Medical

Research is registered with CPCSEA and has a valid registration number

424/RO/c/01/CPCSEA.

M.P. Save et al.80

B: Laboratory investigations:

Slit skin smear examination (SSS): was done from at least three sites by trained paramedical

staff. Slides were stained using Ziehl Neelsen Carbol Fushin (ZNCF), and scored for

bacteriological index (BI).16

Collection of skin/nerve biopsies: Skin/nerve biopsies were obtained from patients after

informed consent and collected as under:

At Registration (onset): Two incision skin biopsies were obtained from all the patients;

(a) Site showing maximum activity/reaction, referred to as Lesion (L) biopsy

(b) Non-lesion area (usually a mirror image site) to serve as control, referred to as

Mirror (M).

In 10 Patients biopsy of an involved cutaneous nerve was obtained under local anesthesia.

The skin overlying the nerve served as the inactive (non- lesion) M site.

During follow up: At the onset of a repeat episode of T1R, and at 3 months post- release from

treatment from those who did not develop any T1R, only the lesion (L) site were biopsied.

Each biopsy was divided in to 3 parts, processed and studied as under

(1) One part was fixed in Formal-Zenker, processed and embedded in paraffin.

1-a Histopathology: Five micron thick paraffin embedded sections stained with Trichrome

modified Fite-Faraco (TRIFF) were examined for acid-fast bacilli (AFB), the characteristics of

the granuloma and used for leprosy classification using the Ridley-Jopling scale.15

1-b Immunohistochemical detection and localization of Ag85: The paraffin embedded

tissue sections were also used as described below.

Paraffin sections of lesional and mirror image biopsies were subjected to indirect

immunoperoxidase staining using rabbit raised polyclonal antibody (kindly supplied by Prof.

Harald Wiker, University of Bergen, Norway) for detection of the antigen. Peroxidase-

conjugated swine anti-rabbit (Dakocytomation) served as a secondary antibody. Endogenous

peroxidase activity was blocked using 3% H2O2 and 1% foetal calf serum was used for non-

specific blocking. The reaction was developed with 3-3’–diaminobenzedine (DAB) as

chromogen substrate and counter-stained with haematoxylin.17

(2) Detection of M. leprae specific 16s rRNA using in situ RT PCR

The Second part of the biopsy collected in 10% buffered formalin was used for the detection

of 16s rRNA, by direct in situ RT-PCR, this work was conducted at National JALMA

Institute for Leprosy and other Mycobacterial Diseases (NJIL & OMD).

In situ reverse transcriptase polymerase chain reaction (in situ RT-PCR) In situ

localisation of M. leprae 16s rRNA was carried out using Fast start Taq DNA polymerase,

Digoxigenin (DIG) labeled nucleotides, anti DIG antibody labeled with alkaline phosphatase

and 5-Bromo-4-Chloro-30-indolyphosphate - Nitro Blue Tetrazolium (BCIP-NBT) as sub-

strate chromogen assisted detection. A dark blue signal in the cells indicated presence of

rRNA, hence of viable M. leprae. Later DAB was preferred; presence of brown precipitate of

DAB in inflammatory macrophages, In Schwann cells in the nerve biopsies (number of nerve

biopsies ¼ 3) and in dermal nerves in skin biopsies was recorded.11,12


(3) Assessment of M. leprae viability using mouse foot pad method (MFP):

The third part of the biopsy was used for MFP study using the standard method. In brief, the

tissue was homogenized with a glass homogenizer within 24 hours of collection. A fixed

volume (i.e. 0·001ml) of the homogenate was uniformly smeared on spot slides, dried and

stained with ZNCF. The number of AFBs was determined using standard protocol outlined by

World Health Organization.13 The bacillary load per gram weight of tissue was determined,

adjusted to 1 £ 104 AFB/30ml of homogenate and injected into both hind foot pads of normal

Swiss white (S/W) mice. Skin homogenates that scored negative for AFB were injected

without further dilution. The bacillary count was counted at 6th, 7th, 8th and 12th months post-

inoculation. A foot pad yield of $ 1 £ 105 was considered positive.

Results

As depicted in Table 1, of the 165 subjects recruited for this study male to female ratio was

2·3:1 and the average age recorded was 32 ^ 14.

CLINICAL

An account of clinical samples examined using different methods is shown in table 2a.

Time Scale for the collection of biopsy specimen at onset as well as re biopsies is depicted

in table 2b.

Frequency of T1R and its recurrence in the subjects: Of the 165 patients recruited for

the study 63 were from T1R (þ ) group. Of these 63, 58 (35%) were seen with T1R at

registration of which majority had skin only reaction (39 ¼ 67%), 16 (27%) had skin þ

nerve reaction and three had only nerve reaction. In the remaining, five (3%) cases, a 1st

episode of T1R was recorded within the first 3 months of starting MDT. Among the 58

cases presenting with T1R, 40 (69%) had single episode and 18 (31%) had multiple (2 or

more) episodes of T1R. In the five cases that developed an incidence of T1R; two had

multiple episodes. Thus in the T1R (þ ), 20 (32%) had more than one episode of T1R during

the course of this study (Table 3). Whereas, in T1R (2 ) (n ¼ 102), 11 (10%) cases

developed episode of reaction at any time point after 3 months, of which one had two

episodes.

LABORATORY INVESTIGATIONS

Biopsy figures and results are depicted in Tables 4, 5a, 5b and 6.

Table 1. Gender and average age of the patients under study

Variables No.

Total no cases 165Male patients 115Female patients 50Male to Female ratio 2·3:1Average Age & SD 32 yrs ^ 14

M.P. Save et al.82

I. Histopathological Classification (Ridley-Jopling) and frequency of T1R in the different

leprosy types

Of the 165 lesion biopsies studied from 165 patients, histopathological features of BT leprosy

were seen in 112 (68%) mid-borderline (BB) in 13 (8%) and BL type of leprosy in 40 (24%)

of cases. Frequency of T1R was highest among BB (69%) followed by BT (36%) and BL

(35%) (Table 4).

Further, all the cases with clinical evidence of T1R also had histopathological evidence

of T1R (n ¼ 65). In addition, 18 cases (11%) had only histopathological evidence of T1R

(sub-clinical).

Among the re-biopsies, 19 (68%) were BT leprosy, three were mid-borderline (BB) and

six were BL type of leprosy (Table 8).

II. Bacteriological findings in relation to T1R (Table 5a)

All the 165 cases were scored for the presence of AFB in tissue homogenates and/or in tissue

sections (histopathology), 31(19%) scored positive and others (n ¼ 134) were negative.

Of the 31 AFB positive in ‘L’, 16 (52%) were T1R (þ ) and 15 (48%) were T1R (2 ).

Of the 134 AFB negative patients, 47 (35%) were T1R (þ ) and 87 (65%) were T1R (2 ).

Among the 28 re-biopsies, three scored positive for AFB; all three had repeat episodes of

T1R and were from T1R (þ ) group.

III. Immunohistochemical detection and localization of Ag 85: (Figures 1 & 2)

(a) As compared to AFB scores, percentages of both ‘L’ and ‘M’ scoring positive with

Antigen 85 are significantly higher in all three types of leprosy i.e. BT, BB and BL, proving

its higher sensitivity. (b) Positive scores with Ag 85 are significantly higher in ‘L’ as

compared to ‘M’ particularly in BT lesions c) Overall positive scores (%) is higher in T1R(þ )

(74%) group as compared to T1R (2 ) (66%) but the difference is not statistically significant

(P ¼ 0·6). (Table 5a)

Table 2a. Account of number of clinical samples examined at onset and re-biopsies using different methods

Test Lesion (onset) Mirror (onset) Lesion (rebiopsies) Mirror (rebiopsies)

Histopathology 162 162 28 22MFP 158 141 28 21Ag 85 detection 76 73 23 1816s RNA- in situ RT PCR 28 28 9 8

Table 2b. Time Scale of biopsies collected at onset and re-biopsies in T1R (þ ) and T1R (2) cases

Onset 3–6 m 7–9 m 10–12 m .12 m

T1R (þ) 63 7 2 4 2T1R (2) 102 0 0 0 13


A semi-quantitative assessment of Ag 85 positivity is depicted in Table 6. Notably, 20/38

(52%) L biopsies from T1R (þ ) cases scored $ 2þ and 11/20 (55%) were BT-BB cases,

indicating that intensity of staining is disproportionately higher among the BT lesions

showing T1R.

Localization of Ag85:

Localization of Ag 85 staining was restricted to areas where M. leprae could be found viz.

strong antigen 85 positivity was seen in dermal nerves mainly in the Schwann cells and in the

inflammatory cells such as macrophages (Figure 3). Occasionally muscle spindles in the skin

also scored positive for Ag 85.

An account of antigen 85 positivity recorded in the skin/dermal nerves:

Skin lesions from 26 patients scored positive for Ag 85 in the dermal nerves. Notably 19

among them (73%) were with T1R (þ ), showing a strong association between presence of

Ag 85 within the dermal nerve and T1R.

A similar positive association was also seen in the re–biopsies. Time scale of the re-

biopsy is given in Table 2b. Of the 23 re-biopsies (all lesions), including 13 with T1R (þ ) and

10 without T1R (2 ), the proportion of cases with positivity in T1R (þ ) was 8/13 (62%), four

among them were BT cases and T1R (2 ) was 5/10 (50%), was not statistically significant.

Overall, 13/23 (57%) cases were Ag 85 þve, indicating presence of viable bacilli among

cases receiving 6–12 months of MB-MDT (Table 7).

IV. In situ reverse transcriptase polymerase chain reaction (in situ RT-PCR) (Figures 4 & 5)

(a) Overall, the proportion of cases scoring positive in the 16s rRNA detection method

was higher than Ag85 in both ‘M’ and ‘L’ biopsies indicates higher sensitivity of the

former.

(b) In the lesions the scores were higher in T1R (þ ) group as compared to T1R (2 )

(15/16 (94%) and 10/12(83%) respectively) however the difference was statistically

not significant (Table 5a).

Table 3. Account of T1R at onset, developed within 3 months and its recurrence

Site of Reaction At registration (n ¼ 58) (%) Developed within 3 m (n ¼ 5) Total (n ¼ 63) (%)

Only Skin 39 (67) 5 44 (70)Skin1Nerve 16 (27) 0 16 (25)Only nerve 3 (5) 0 3 (5)No of reaction episodesSingle 40 (69) 3 43 (68)$2 episodes 18 (31) 2 20 (32)

Table 4. Overall frequency of T1R and leprosy class at onset (RidleyJopling, histopathological scale)

Variables BT BB BL Total (%)

T1R (þ ) 40 (36%) 9 (69%) 14 (35%) 63 (38%)T1R (2) 72 4 26 102Total 112 13 40 165

M.P. Save et al.84

(c) In the re-biopsies, all 4 from T1R (þ ) and 3/5 from T1R (2 ) scored positive for

M. leprae specific 16s rRNA by in situ RT-PCR, further ascertaining the presence of

viable bacteria in patients on treatment (MB-MDT) or having completed treatment

(Table 7).

V. Viability of M. leprae assessed through Mouse foot pad test

Mouse foot pad test results of 158 Lesion (L) & 141 Mirror (M) biopsies under different

leprosy type are depicted in Table 5. The former includes 11 nerve biopsies. Overall, 74 (46%)

Table 5a. Results of lab tests i.e. AFB, Ag85, 16s rRNA and MFP in relation to T1R(þ ) and T1R(2) in differentleprosy class expressed as No. positive/No. tested in the Mirror (M) and lesion (L) sites

Leprosy Class

Groups Biopsy site BT (%) BB (%) BL (%) Total (%)

AFB detection (in Homogenate or histopathology)T1R (þ) M 1/37 0/8 2/13 3/58 (5)

L 3/40 (7) 2/9 (22) 11/14 (79) 16/63 (25)T1R (2) M 0/62 0/4 1/20 1/86 (1)

L 2/72 (3) 0/4 13/26 (50) 15/102 (15)Ag85 detection

T1R (þ) M 4/20 (20) 1/7 (14) 5/10 (50) 10/37 (27)L 13/21 (62) 5/7 (71) 10/10 28/38 (74)

T1R (2) M 5/23 (22) 1/3 6/10 (60) 12/36 (34)L 14/25 (56) 2/3 9/10 (90) 25/38 (66)

16s r RNA- detection (In situ RT-PCR)T1R (þ) M 5/9 0/1 4/6 9/16 (56)

L 8/9 1/1 6/6 15/16 (94)T1R (2) M 3/8 – 3/4 6/12 (50)

L 6/8 – 4/4 10/12 (83)Growth in the Mouse Foot Pad

T1R (þ) M 0/35 0/8 2/13 2/56 (4)L 24/38 (63)a 3/8 13/14 (93) 40/60 (66)b

T1R (2) M 0/60 0/4 4/19 4/83 (5)L 16/70 (23)a 1/4 17/24 (71) 34/98 (35)b

Key: T1R (þ ) - reaction group, T1R (2) - no reaction group, M – Mirror biopsy, L – Lesion biopsy, BT-Borderline tuberculoid, BB- Borderline, BL- Borderline lepromatous, AFB- Acid fast bacilli, 16s rRNA- In situRT-PCR (In situ reverse transcriptase polymerase chain reaction)

a – p valve: 0·00008 [OR ¼ 5·7, RR: 2·7 (95%Cl)] (between T1R (þ ) and T1R (2) group [BT class])b – p valve: 0·00018 [OR ¼ 0·27, RR: 0·52 (95%Cl)] (between T1R (þ) and T1R (2) group [total values])

Table 5b. Comparative analysis of number of cases positive for 2 (i.e. Ag85 and MFP) and 3 (i.e. Ag85, 16s rRNAand MFP) tests in smear þve and smear 2ve T1R (þ) and T1R(2) groups at onset in select cases

Smear þve Smear 2ve

2 Tests þveAll 3

Tests þveAll 2 or 3Test 2ve 2 Tests þve

All 3Tests þve

All 2or 3Test 2ve Total þve

GrandTotal

T1R (þ) 8 11 0 1 2 4 (15%) 22 (85%) 26T1R (2) 3 2 0 3 4 8 (40%) 12 (60%) 20


of L [40 in T1R (þ ) and 34 in T1R (2 )] & six (4%) of M scored positive in the MFP test. All

six M biopsies that scored positive were from BL cases. A significantly higher proportion of L

biopsies from T1R (þ ) patients tested positive in the MFP test, confirming the presence of

viable M. leprae 40/60 (66%) as compared to T1R (2 ) 34/98 (35%) and this difference is

statistically highly significant (P ¼ 0·00018) (Table 5a).

MFP FINDINGS IN AFB – VE CASES

Among the 74 MFPþve cases in ‘L’ biopsies, 24/40 (60%) in T1R (þ) and 24/34 (70%) in T1R

(2) were AFB –ve in the tissue homogenates. Majority were BT-BB type of lesion. Four cases

in T1R (2) which were AFB –ve and MFPþve developed an episode of reaction at a time point

after 3m and were BT-BB. Overall, MFP positivity among the AFB –ve biopsies was 24/42

(51%) in T1R (þ) and 24/87 (27%) in T1R (2) which was also significant (P ¼ 0·01).

A small number of mirror (M) biopsies from both the groups i.e. T1R (þ ) two (3%) and

T1R (2 ) four (5%) respectively scored positive in the MFP and difference was not

significant.

None of the 28 re-biopsies, including 15 T1R (þ ) and 13 T1R (2 ) cases subjected to

MFP test showed any fold increase/growth in MFP (Table 7).

Comparative analysis of all three tests viz growth in MFP, detection of Ag85 and

16srRNA, wherever done, in smear þve and smear –ve patients, reveal that proportion

scoring all tests negative is higher in the T1R (2 ) [8/20 ¼ 40%] as compared to T1R (þ )

[4/26 ¼ 15%]. While a positive result is an indicator of, or in case MFP test confirms the

presence of viable bacteria, a negative finding does not rule out the presence of viable

bacteria, considering that a small part of biopsy specimen was tested. (Table 5b)

To summarise, at onset while the MFP test showed a highly significant difference in the

positive score between the two groups, whereas in the other two tests i.e. Ag 85 and 16s

rRNA, the overall proportion of positive scores was higher in T1R (þ ) group but the

difference was statistically not significant. Thus antigen based tests displayed lower

discriminatory power. In case of repeat biopsies, however the antigen based tests showed a

better discriminatory power while mouse foot pad test was of no value.

Table 6. Semi-quantitative assessment scores of Ag 85 in L biopsies in relation to T1R (þ ) and T1R (þ) and leprosyclass (Ridley- Jopling)

T1R (2) T1R (þ)

Ag 85 grades BT BB BL Total BT BB BL Total

11 8 2 4 14 5 2 1 821 6 0 1 7 7 2 3 1231 0 0 4 4 1 1 5 741 0 0 0 0 0 0 1 1Total 14 2 9 25 13 5 10 38

11/25 (44%) L biopsies from T1R (2) cases scored $ 2 þ and 6 among them were BT-BB20/38 (52%) L biopsies from T1R (þ ) cases scored $ 2 þ and 11 among them were BT-BBGrades: 1þ Trace positivity, localized in few cells

2þ Localised but many cells þve/dispersed but few cells/location3þ Dispersed positivity with many cells/location4þ Majority of the cells positive

M.P. Save et al.86

Discussion

In leprosy around 25–50% of borderline group of patients (BT to BL) experience Type 1

Lepra reaction during the course the disease before the initiation of treatment, during and

even after the completion of MDT and is the commonest cause of morbidity.3,5 Reaction and

ensuing nerve damage is managed by the use of immunosuppressive/anti inflammatory drugs

such as corticosteroids.2 However, the important question of what precipitates such a

reaction, which is key to its prevention/prediction and management remain unclear.

The present study is built around a working hypothesis viz; viable M. leprae play an

important role in the precipitation of Type 1 reaction in leprosy. To demonstrate the presence

of viable M. leprae in a reacting lesion, three tests were applied concurrently viz. growth in

Figures 1 and 2. Skin lesion biopsies from a BB case stained with Antigen 85 antibody. Note positive but weak Ag 85staining at onset (Figure 1) with out T1R vs the a re-biopsy obtained during an episode of T1R (Figure 2) showinghigher intensity of staining (arrow).


the mouse foot pad, immunohistochemical detection of M. leprae secretory protein Ag 85,

and detection of M. leprae specific 16s rRNA – using in situ RT-PCR.

It is important to state the known limitations in the use of these methods viz:

(1) viable bacteria are present in the normal course of the untreated disease

(2) none of the test/s quantifies the number of viable bacteria in a given sample

(3) none of these tests by itself or in combination fulfill the desired sensitivity and

specificity

Taking these limitations into account, our first assumption is, since T1R is an immune

mediated Delayed-Type Hypersensitivity (DTH) type of reaction mounted by the host against

bacteria there should be higher /better killing of bacteria at the site of reaction which in turn

should result in decrease in number of viable bacteria. It is anticipated therefore, overall

positive score (%) should be lower in T1R (þ ) lesions/cases, in a given test denoting

viability.

Our second assumption is, if there is a higher positive score during or before a clinical

reaction and its persistence during a relapse, this could be a good indication of their

involvement in the process.

Figure 3. Detection of antigen 85 in the dermal nerve in a skin lesion from a BT patient with T1R at onset.

Table 7. Results of various test in re biopsies i.e. detection of Ag85, 16s rRNA- In situ RT-PCR and MFP methods inLesion (L) and Mirror (M) expressed as No. positive/No. tested among T1R (þ) and T1R (2) groups

Lesional re-biopsy Mirror re-biopsy

T1R (þ) T1R (2) Total T1R (þ ) T1R (2) Total

Ag85 positive 8/13(62) 5/10 (50) 13/23 (57) 3/9 (33) 3/9 (33) 6/18 (33)16s RNA– in situ PCR 4/4 3/5 7/9 1/3 1/5 2/8 (37)Viability using MFP test 0/15 0/13 0/28 0/10 0/11 0/21

Key: T1R (þ ) - reaction group, T1R (2) - no reaction group, 16s rRNA- In situ RT-PCR - In situ reversetranscriptase polymerase chain reaction.

M.P. Save et al.88

In this study, comparison is between a reaction lesion (skin or nerve) T1R (þ ) with no

reaction T1R (2 ) lesions. Only patients presenting with or developing clinical signs of T1R

within the first 3 months of initiation of MDT in the skin with or without neuritis are

considered as T1R (þ ) cases.

As anticipated, presence of viable bacteria, were demonstrated in both T1R (þ ) and T1R

(2 ) group of patients. However, there was a marked difference in the quality or metabolic

state of M. leprae detected in the two groups was evident from the following three findings;

(1) A significantly higher proportion of lesion biopsy homogenates obtained at onset, from

T1R (þ ) cases scored positive (have shown unequivocal growth in MFP test, proving

the presence of viable bacteria), as compared to T1R (2 ) (P , 0·005). Also, MFP

positivity among AFB –ve L biopsies was significantly higher in T1R (þ ) (P , 0·05).

In contrast, a small proportion of non lesion biopsies (mirror site) scored positive in

both T1R (þ ) and T1R (2 ) group and the difference was not statistically significant

Figure 4. Demonstration of M. leprae specific 16 s rRNA using in situ RT PCR, BT skin lesion collected during 2nd

episode of T1R at 6 months. Dermal nerve showing positive signal (arrow).

Figure 5. Skin lesion from a BT case, collected during 2nd episode of T1R at 13 months. M. leprae specific 16s rRNApositive signal (arrow) is seen in a small dermal vessel.


(2) With respect to Ag85, while the overall positive scores were higher in the T1R (þ )

group but the difference was not statistically significant, the intensity of staining (grade

$ 2þ ) was disproportionately higher in BT – BB lesions with T1R (þ )

(11/20 ¼ 55%).

(3) In the re biopsies obtained during a repeat episode of T1R, Ag 85 as well as 16s rRNA,

positive scores (%) were higher in theT1R (þ ) lesions.

It can be inferred therefore ‘viable’ bacteria are an essential component and probably play

a crucial role in Type 1 reaction.

Conversely, difference in the quality of bacilli and not the quantity or the ratio of dead to

viable; play a role in the precipitation of Type 1 reaction in a given site.

Supportive evidence for the above statement also comes from a preliminary study finding,

using real time PCR (results not shown). A higher cDNA copy numbers of tlyA gene

(indicator of metabolically active M. leprae) in lesion (L) biopsies from T1R (þ ) as

compared to T1R (2 ) cases were detected.

Another study using real time analysis demonstrates presence of significant amount of

mRNA for the hsp18 gene, in reaction lesions indicating the existence of live bacilli in

reversal reaction cases.18 Other documented evidence viz (a) higher incidence of viable

M. leprae in cases with sub-clinical (histopathological) evidence of T1R as compared to T1R

(2 ) cases.8,19 (b) higher occurrence of viable M. leprae among patients with late onset

reaction are also in support.20 Further, bacteriologically positive patients are 3·2 times at a

higher risk of developing Type 1 reaction is in consonance with the earlier documented

findings.21 One of the widely accepted views is that, M. leprae products (antigens) are

involved in the precipitation of T1R. It is believed, M. leprae antigens such as PGL-1 and

18 kDa antigen (stress protein) may be involved in the precipitation of T1R.22,23 Autoimmune

response to bacillary antigens as a cause for T1R precipitation was suggested by Naafs.24 As

opposed to these our study demonstrated the presence/involvement of metabolically active

M. leprae in the precipitation of T1R.

Notably in the re-biopsies collected during or on completion of treatment, all four from

T1R (þ ) and 3/5 from T1R (2 ) scored positive for M. leprae specific 16s rRNA by in situ

RT-PCR, ascertaining the presence of viable bacteria. Proportion of cases scoring positive

with antigen 85 were higher in T1R (þ ) lesions. None scored positive in the MFP test is

attributable to its limitation as a test.

Besides the strong association seen between viable bacteria and Type 1 reaction,

detection of Ag 85 positivity in 13/23 (57%) and M. leprae specific 16s r RNA in 7/9 (77%) of

re-biopsies, fact that they were patients receiving . 6 months or having completed 12 months

of MB-MDT, imply that M. leprae remain refractory to antileprosy treatment is worrisome.

Table 8. Account of and classification of cases with reaction [T1R (þ)] and without reaction [T1R (2)] in differentleprosy class as per Ridley Jopling scale (histopathological) in rebiopsies

Variables BT BB BL Total cases

T1R (þ ) 9 2 4 15T1R (2) 10 1 2 13Total 19 3 6 28

M.P. Save et al.90

This finding has an important bearing on the management of T1R cases as well as raises

question regarding the efficacy of MDT.

Reactions that occur post release from treatment in around 5–10% of cases, termed as

‘late reversal’ reaction, are generally being treated with corticosteroids alone under the

assumption that these reactions are precipitated by bacterial products (antigens).25,26 Findings

from this study allow us to emphatically state that any long term treatment with steroids must

be covered with anti-leprosy treatment.

Not surprisingly, a strong association was seen between T1R and the localisation,

increased frequency as well as intensity of antigen 85 staining in the dermal nerves. The

Schwann cells in particular are a known reservoir of M. leprae.27 It is conceivable therefore,

survival of M. leprae within the Schwann cells/nerves, increases the vulnerability of patients

towards reaction and neuritis.

This study using three viability assessment methods viz. detection of M. leprae secretory

protein i.e. Ag 85, detection of M. leprae specific 16s r RNA using in situ RT PCR and growth

in MFP, firstly demonstrates the presence of viable M. leprae in the reaction lesions in a

significantly higher proportion of cases in the mouse foot pad test that is the gold standard,

confirming the presence of viable M leprae, are an essential component of T1R, thus

strengthening our hypothesis. Importantly, the bacilli are ‘metabolically active’ and results of

the three viability tests complement each other.

We are aware that the three tests used in this study have no practical value in predicting or

diagnosing T1R.

Preliminary data (results not shown) show over-expression of M. leprae gene associated

with growth and metabolic activity viz tlyA gene in real time PCR. This is in line with the

positive association seen between metabolically active M. leprae and T1R. Study using

Expression analysis of genes related to metabolism and virulence of M. leprae from human

host by micro-array has shown over expression of accA3 gene in patients with reaction.28

These certainly have the potential to be developed as a marker for predicting T1R.

In conclusion, our study findings show that ‘metabolically active’ M. leprae is a

component/prerequisite and the secretory protein Ag 85, might be the trigger for precipitation

of T1R.

Acknowledgements

We would like to thank Indian Council for Medical Research for the financial support

(sanction no: 5/8/3(3)2005-ECD-1). Our Collaborators: Bombay Leprosy Project, Mumbai

and Kustharog Nivaran Samiti, Shantivan, Panvel for referral of patients and their field staff

in patient recruitment and patient holding. Dr. Sunil Ghate (Clinician) for examination,

documentation and management of patients. Mr Ramchandra Chile for Histopathology

preparation and staining.

We acknowledge Dr. Kiran Katoch, Director National JALMA Institute for Leprosy and

other Mycobacterial Diseases (NJIL & OMD) for facilitating this study and transfer of

technology from NJIL to FMR. Staff and students of department of microbiology and

pathology for demonstration of molecular biology techniques involving mRNA detection.

Dr. Nerges Mistry, Director, FMR for her all round support and valuable suggestions and

Dr Shubhda Pandya for helping us with preparation of the manuscript. Finally, all the patients

and their family members for participating in the study.


References

1 Van Brakel WH, Khawas IB, Lucas SB. Reactions in leprosy: an epidemiological study of 386 patients inWest Nepal. Lepr Rev, 1994; 65: 190–203.

2 WHO expert committee on leprosy, WHO technical report series 1998; 874.3 Lockwood DNJ. Steroids in leprosy Type I (reversal) reactions: mechanism of action and effectiveness. Lepr Rev,

2000; 71: 111–114.4 Walker SL, Lockwood DNJ. Type 1 reactions and their management. Lepr Rev, 2008; 79: 372–386.5 Lockwood DN, Vinayakumar S, Stanley JN, McAdam KP, Colston MJ. Clinical features and outcome of reversal

(Type 1) reactions in Hyderabad, India. Int J Lepr Other Mycobact Dis, 1993; 61: 8–15.6 Roche PM, LeMaster J, Butlin RC. Risk factors for type I reaction in leprosy. Int J Lepr Other Mycobact Dis,

1997; 65: 450–455.7 Sengupta U. Immunopathology of leprosy-current status. Ind J lepr, 2000; 72: 381–391.8 Shetty VP, Wakade A, Antia NH. A high incidence of viable Mycobacterium leprae in post MDT recurrent lesions

in tuberculoid leprosy patients. Lepr Rev, 2001; 72: 337–344.9 Rambukkana A, Das PK, Krieg S, Faber WR. Association of the mycobacterial 30-kDa region protein with the

cutaneous infiltrates of leprosy patients. Evidence for the involvement of major mycobacterial secreted proteins inthe local immune response of leprosy. Scan J Immunol, 1992; 36: 35–48.

10 Launois P, Niang MN, Sarthou JL et al. T-cell stimulation with purified mycobacterial antigens in patients andhealthy subjects infected with Mycobacterium leprae: secreted antigen 85 is another immunodominant antigen.Scand J Immunol, 1993; 38: 167–176.

11 Kurabachew M, Wondimu A, Ryon JJ. Reverse transcription PCR detection of Mycobacterium leprae in clinicalspecimens. J Clin Microbiol, 1998; 36: 1352–1356.

12 Hirawati Katoch K, Chauhan DS et al. Detection of M. leprae by reverse transcription PCR in biopsy specimensfrom leprosy patients. J Commun Dis, 2006; 38: 280–287.

13 WHO document, Mouse foot pad techniques. pp: 63–66, In Laboratory techniques of leprosy; WHO/CDS/Lep 86.4.

14 Snedecor GW and Cochran WG. Statistical Methods, Calcutta: Oxford and IBD Publishing Co. 1968.15 Ridley DS, Jopling WH. Classification of leprosy according to immunity. Five group system. Int J Lepr Other

Mycobact Dis, 1966; 34: 255–273.16 Wheeler EA, Hamilton EG, Harman DJ. An improved technique for the histopathological diagnosis and

classification of leprosy. Lepr Rev, 1965; 36: 37–39.17 Sternberger LA, Hardy PH, Cuculis JJ, Major HG. The unlabelled antibody enzyme method of

immunohistochemistry. Preparation and properties of soluable antigen antibody complex (horseradish peroxidaseantihorse raddish peroxidase) and its use in the indentification of spirochetes. J Histochem cytochem, 1970;18: 315.

18 Lini N, Shankernarayan NP, Dharmalingam K. Quantitative real-time PCR analysis of Mycobacterium lepraeDNA and mRNA in human biopsy material from leprosy and reactional cases. Journ of Med Micro, 2009; 58:753–759.

19 Shetty VP, Wakade AV, Ghate SD et al. Clinical, histopathological and bacteriological study of 52 referral MBcases relapsing after MDT. Lepr Rev, 2005; 76: 241–252.

20 Shetty VP, Khambati FA, Ghate SD et al. The effect of corticosteroids usage on the bacterial killing, clearance andnerve damage in leprosy; part 3- study of two comparable groups of 100 multibacillary (MB) patients each, treatedwith MDTþ steroids verses MDT alone, assessed at 6 months post release from 12 months MB-MDT. Lepr Rev,2010; 81: 41–58.

21 Ramu G, Desikan KV. Reactions in borderline leprosy. Ind J Lepr, 2002; 74: 115–127.22 Stefani MM, Martelli CM, Morais-Neto OL et al. Assessment of anti PGL-1 as a prognostic marker for leprosy

reactions. Int J Lepr Other Mycobact Dis, 1998; 66: 356–364.23 Mohanty KK, Joshi B, Katoch K, Sengupta U. Leprosy reactions: Humoral and cellular immune responses to

M. leprae 65 kDa, 28 kDa and 18 kDa antigens. Int J Lepr Other Mycobact Dis, 2004; 72: 149–158.24 Naafs B. Current views on reactions in leprosy. Int J Lepr Other Mycobact Dis, 2000; 72: 97–122.25 Pannikar V, Jesudasan K, Vijayakumaran P, Christian M. Relapse or late reversal reaction. Int J Lepr Other

Mycobact Dis, 1989; 57: 356–364.26 Kumar B, Dogra S, Kaur I. Epidemiological characteristics of leprosy reactions: 15 year experience from north

India. Int J Lepr Other Mycobact Dis, 2004; 72: 125–133.27 Shetty VP, Suchitra K, Uplekar MW, Antia NH. Higher incidence of viable Mycobacterium leprae with in nerve

as compared to skin among multibacillary leprosy patients released from multidrug therapy. Lepr Rev, 1997; 68:131–138.

28 Sharma R, Lavania M, Chavan DS et al. Potential of a metabolic gene (accA3) of M. leprae as a marker for leprosyreactions. Indian J Lepr, 2009; 81: 141–148.

M.P. Save et al.92

78 Association of viable Mycobacterium leprae with Type 1 reaction ...

Documents

Transcript of 78 Association of viable Mycobacterium leprae with Type 1 reaction ...